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OPEN
Received: 23 September 2016 Accepted: 25 April 2017 Published: xx xx xxxx
High-dose maternal folic acid supplementation before conception impairs reversal learning in offspring mice Kristin S. Henzel1, Devon P. Ryan 1,3, Susanne Schröder1, Marco Weiergräber2 & Dan Ehninger1 Maternal folic acid (FA) supplementation prior to and during gestation is recommended for the prevention of neural tube closure defects in the developing embryo. Prior studies, however, suggested that excessive FA supplementation during gestation can be associated with toxic effects on the developing organism. Here, we address whether maternal dietary folic acid supplementation at 40 mg/ kg chow (FD), restricted to a period prior to conception, affects neurobehavioural development in the offspring generation. Detailed behavioural analyses showed reversal learning impairments in the Morris water maze in offspring derived from dams exposed to FD prior to conceiving. Furthermore, offspring of FD dams showed minor and transient gene expression differences relative to controls. Our data suggest that temporary exposure of female germ cells to FD is sufficient to cause impaired cognitive flexibility in the subsequent generation. It has been known for decades that folic acid (FA) supplementation before and during pregnancy reduces the incidence of neural tube defects (NTDs) in newborns1, 2. Many countries have therefore established national programs for mandatory FA fortification of food, which has reduced the occurrence of NTDs by up to 46%3–5. It has also been recommended that women who might become pregnant should take daily FA supplements to minimise the risk of NTDs6. Since the first fortification programs started, global FA intake has increased dramatically. In the United States, for example, this increase was twice as large as anticipated prior to the implementation of fortification programs and, consequently, serum folate concentrations on a population level may be up to 150% higher than in the 1990 s7, 8. While the population-level benefits of FA supplementation are well documented, some previous studies have noted possible adverse consequences of certain FA supplementation regimens. In rat offspring, a change in body weight and metabolism as well as an increased risk for mammary adenocarcinomas are associated with high maternal folate levels9–11. In mice, disrupted fetal development, embryonic loss, and embryonic developmental delay can be caused by a high-dose FA supplementation in the maternal food during gestation12, 13. In humans, the risk to develop allergies, atopic dermatitis and childhood asthma is increased if the mother consumes large amounts of FA during pregnancy14–17. In a previous study in mice we showed that paternal consumption of a methyl donor-rich diet, including elevated concentrations of folic acid, had effects on cognitive and neural functions in offspring mice18. Elevated folate levels have also been linked to epigenetic changes. In vitro, gene expression levels shift as a function of the extracellular folate concentration19. Maternal folate status in mice has an impact on DNA methylation and gene expression in the offspring’s cerebral hemispheres and at specific loci in the fetal gut20–22. High maternal FA also influences gene expression in the cerebellum of mouse pups23. Global gene methylation decreases in coagulated blood samples of women after taking daily FA supplements24 and children of supplemented mothers show increased IGF2 methylation25. 1
German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Straße 27, 53127 Bonn, Germany. Research Group Experimental Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany. 3Present address: Max Planck Institute for Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany. Correspondence and requests for materials should be addressed to D.E. (email: [email protected]) 2
Scientific Reports | 7: 3098 | DOI:10.1038/s41598-017-03158-1
1
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Figure 1. Schematic illustration of the study design.
There are still many unresolved questions concerning possible health consequences of high FA intake. In particular, the impact of maternal FA supplementation on cognitive and behavioural abilities in the offspring remains insufficiently investigated. Moreover, the studies available mostly involve designs with direct exposure of the developing offspring to excessive folate during the entire period of in utero development and during early postnatal development. Thus, behavioural and epigenetic effects could be direct consequences of high folate doses upon the developing individual. To address whether dietary FA supplementation prior to conception has effects on the offspring generation, we restricted maternal FA supplementation to a period prior to mating (see Fig. 1 for schematic illustration of study design). Under these conditions, there is exposure of the female, including her germ cells, to high FA doses, while direct gestational effects on the developing organism are limited because excess folate is eliminated rapidly26–28. In an earlier study12 it was shown that folic acid supplementation at 40 mg/kg diet fed to dams before and during pregnancy caused embryonic defects and developmental delay. We based our FA supplementation regimen on this prior work12 and wanted to assess possible effects of this diet on cognition, behaviour and hippocampal gene expression in the offspring of FA-supplemented dams. Our studies revealed clear deficiencies in reversal learning caused by an FA-rich diet fed before mating. As FA supplementation shortly before and during pregnancy has been widely recommended and adopted29, these results may provide important insights regarding possible adverse intergenerational effects associated with excessive folate intake that should be studied in more detail in human populations.
Results
Spatial learning and reversal learning in the Morris water maze. We examined spatial learning and memory in CD and FD F1 mice using a hidden version of the Morris water maze. Analysis of escape latencies during the first seven days of training showed comparable acquisition by CD and FD F1 mice (Fig. 2). There was a significant effect of day on escape latencies (two-way ANOVA: F (6, 252) = 23.35, p
OPEN
Received: 23 September 2016 Accepted: 25 April 2017 Published: xx xx xxxx
High-dose maternal folic acid supplementation before conception impairs reversal learning in offspring mice Kristin S. Henzel1, Devon P. Ryan 1,3, Susanne Schröder1, Marco Weiergräber2 & Dan Ehninger1 Maternal folic acid (FA) supplementation prior to and during gestation is recommended for the prevention of neural tube closure defects in the developing embryo. Prior studies, however, suggested that excessive FA supplementation during gestation can be associated with toxic effects on the developing organism. Here, we address whether maternal dietary folic acid supplementation at 40 mg/ kg chow (FD), restricted to a period prior to conception, affects neurobehavioural development in the offspring generation. Detailed behavioural analyses showed reversal learning impairments in the Morris water maze in offspring derived from dams exposed to FD prior to conceiving. Furthermore, offspring of FD dams showed minor and transient gene expression differences relative to controls. Our data suggest that temporary exposure of female germ cells to FD is sufficient to cause impaired cognitive flexibility in the subsequent generation. It has been known for decades that folic acid (FA) supplementation before and during pregnancy reduces the incidence of neural tube defects (NTDs) in newborns1, 2. Many countries have therefore established national programs for mandatory FA fortification of food, which has reduced the occurrence of NTDs by up to 46%3–5. It has also been recommended that women who might become pregnant should take daily FA supplements to minimise the risk of NTDs6. Since the first fortification programs started, global FA intake has increased dramatically. In the United States, for example, this increase was twice as large as anticipated prior to the implementation of fortification programs and, consequently, serum folate concentrations on a population level may be up to 150% higher than in the 1990 s7, 8. While the population-level benefits of FA supplementation are well documented, some previous studies have noted possible adverse consequences of certain FA supplementation regimens. In rat offspring, a change in body weight and metabolism as well as an increased risk for mammary adenocarcinomas are associated with high maternal folate levels9–11. In mice, disrupted fetal development, embryonic loss, and embryonic developmental delay can be caused by a high-dose FA supplementation in the maternal food during gestation12, 13. In humans, the risk to develop allergies, atopic dermatitis and childhood asthma is increased if the mother consumes large amounts of FA during pregnancy14–17. In a previous study in mice we showed that paternal consumption of a methyl donor-rich diet, including elevated concentrations of folic acid, had effects on cognitive and neural functions in offspring mice18. Elevated folate levels have also been linked to epigenetic changes. In vitro, gene expression levels shift as a function of the extracellular folate concentration19. Maternal folate status in mice has an impact on DNA methylation and gene expression in the offspring’s cerebral hemispheres and at specific loci in the fetal gut20–22. High maternal FA also influences gene expression in the cerebellum of mouse pups23. Global gene methylation decreases in coagulated blood samples of women after taking daily FA supplements24 and children of supplemented mothers show increased IGF2 methylation25. 1
German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Straße 27, 53127 Bonn, Germany. Research Group Experimental Neuropsychopharmacology, Federal Institute for Drugs and Medical Devices, Kurt-Georg-Kiesinger-Allee 3, 53175 Bonn, Germany. 3Present address: Max Planck Institute for Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg, Germany. Correspondence and requests for materials should be addressed to D.E. (email: [email protected]) 2
Scientific Reports | 7: 3098 | DOI:10.1038/s41598-017-03158-1
1
www.nature.com/scientificreports/
Figure 1. Schematic illustration of the study design.
There are still many unresolved questions concerning possible health consequences of high FA intake. In particular, the impact of maternal FA supplementation on cognitive and behavioural abilities in the offspring remains insufficiently investigated. Moreover, the studies available mostly involve designs with direct exposure of the developing offspring to excessive folate during the entire period of in utero development and during early postnatal development. Thus, behavioural and epigenetic effects could be direct consequences of high folate doses upon the developing individual. To address whether dietary FA supplementation prior to conception has effects on the offspring generation, we restricted maternal FA supplementation to a period prior to mating (see Fig. 1 for schematic illustration of study design). Under these conditions, there is exposure of the female, including her germ cells, to high FA doses, while direct gestational effects on the developing organism are limited because excess folate is eliminated rapidly26–28. In an earlier study12 it was shown that folic acid supplementation at 40 mg/kg diet fed to dams before and during pregnancy caused embryonic defects and developmental delay. We based our FA supplementation regimen on this prior work12 and wanted to assess possible effects of this diet on cognition, behaviour and hippocampal gene expression in the offspring of FA-supplemented dams. Our studies revealed clear deficiencies in reversal learning caused by an FA-rich diet fed before mating. As FA supplementation shortly before and during pregnancy has been widely recommended and adopted29, these results may provide important insights regarding possible adverse intergenerational effects associated with excessive folate intake that should be studied in more detail in human populations.
Results
Spatial learning and reversal learning in the Morris water maze. We examined spatial learning and memory in CD and FD F1 mice using a hidden version of the Morris water maze. Analysis of escape latencies during the first seven days of training showed comparable acquisition by CD and FD F1 mice (Fig. 2). There was a significant effect of day on escape latencies (two-way ANOVA: F (6, 252) = 23.35, p
